PROJECT SUMMARY Multiple Sclerosis (MS) is the most common neurological disease of early adulthood and is mediated by autoimmune mechanisms that lead to demyelination and neuronal damage in the central nervous system, resulting in progressive neurological dysfunction. There is no cure for the disease and current treatments focus on preventing future immunological attacks, mainly by suppressing the immune system. This leads to adverse side effects that are often severe or fatal. Accordingly, there is a clear unmet need for the development of effective and well-tolerated therapies to arrest MS development. This has been challenging because MS has numerous etiologies and the molecular mechanisms underlying these etiologies are not well understood. We uncovered the molecular underpinnings of an MS etiology and hope this knowledge will translate into a targeted therapy for MS. This specific etiology results from up-regulation of the soluble form of the Interleukin- 7 Receptor (sIL7R), which has been shown to aggravate the progression and severity of the disease in the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of MS, and to be elevated in patients of several autoimmune diseases including MS, Type I diabetes, Rheumatoid arthritis and Systemic lupus erythematosus. Given that sIL7R is produced by abnormal exclusion of exon 6 from IL7R pre-mRNAs, we developed a novel biologic drug, a splicing-modulating antisense oligonucleotide (SM-ASO; IL7R-005) that corrects this abnormal splicing and restores normal expression of IL7R protein isoforms. IL7R-005 represents a major improvement over current MS therapies in that by correcting IL7R splicing, it diminishes expression of the pathogenic sIL7R isoform, without affecting the function of the membrane-bound IL7R (mIL7R), which is vital for proper immune function, thereby avoiding the adverse immunosuppressive effects of current drugs. T cells are the major producers of sIL7R in humans, and thus to reduce sIL7R levels, IL7R-005 needs to be delivered into T cells in vivo. Although SM-ASOs have been shown to modulate RNA splicing decisions in many tissues in vivo (e.g. FDA-approved Spinraza), the delivery and functionality of SM-ASOs in T cells have not been thoroughly examined, and represents the major hurdle to expand the use of SM-ASOs for treatment of immunological disorders and the development of novel immunotherapies. Here, we address this obstacle by conducting an in-depth, side-by-side analysis of the influence of diverse chemical modifications on the efficiency of SM-ASOs in primary T cells and their potential toxic effects in relevant cell models. This is critical as the chemical modifications of the SM-ASOs could influence their pharmacological properties (e.g., cellular uptake) differentially across cell-types, and thus define the potency of SM-ASOs in a cell-type specific matter. The chemical modifications of the SM-ASOs also dictate potential harmful effects, such as hepatic or renal toxicities. Therefore, this in-depth analysis will enable selection of the chemistry with the optimal therapeutic index (i.e., high potency, low toxicity) for efficient splicing modulation in T cells.